Structural and mechanistic basis of the central energy-converting methyltransferase complex of methanogenesis

Significance Annually, 1 to 2 Gt of the potent greenhouse gas and biofuel methane are produced by methanogenic archaea. A key enzyme of their energy metabolism is a Na+ pumping and vitamin B12-dependent methyltransferase. Here, we present a cryo-EM structure of this multisubunit membrane protein complex that provides the structural information about its unusual architecture and the unique coupling principle between a chemical and an ion-gradient forming process via a vitamin B12 derivative as motor. The energy is provided by two exergonic methyl transfer reactions producing two different Co oxidation and ligation states. It is postulated that the methyl-Co(III) (His-on) state creates an intracellular (inward) entrance and the Co(I) (His-off) state an extracellular (outward) exit for transmembrane Na+ translocation.

methanol or acetate as substrates for performing the final process of anaerobic biomass degradation (1,2).In the hydrogenotrophic variant of methanogenesis (3,4) (shown here) carbon dioxide (CO2) is stepwise reduced to methane (CH4).Reactions 2 -5 are equilibrium reactions.Reaction 8, the exergonic CoM-S-S-CoB reduction to CoM and coenzyme B drives reaction 1, the endergonic CO2 reduction, via a flavin-based electron bifurcation process (5)(6)(7).Reaction 6, the exergonic methyl transfer from methyl-H4MPT to CoM coupled with Na + translocation is catalyzed by MtrABCDEFGH (Mtr, red).The membrane protein carrying the vitamin B12 derivative 5-hydroxybenzimidazolyl cobamide catalyzes the only electrogenic process in the pathway, which is used for ATP synthesis by ATPase (blue).A membrane-spanning [Ni,Fe] hydrogenase (gray) supplies the cycle with additional reduced ferredoxin to maintain a continuous operation when intermediates for the anabolism are withdrawn (3).In the methylotrophic variant (8) methanol, methyl amine and methyl thiol enter the pathway at the methyl-CoM state.From four methyl-CoM three are reduced to methane and one is oxidized backwards to CO2.Acetate and methoxylated aromatic compounds enter their degrading pathways (8,9) at methyl-H4MPT.The methyltransferase reaction is not compulsory for the energy conservation of all methanogenic archaea.Methanomasiliiicoccales e.g.M. stadtmanei growing on methanol, are devoid of Mtr and use an alternative pathway variant (6,10).Methane, the end product of the anaerobic food web, is either anaerobically or aerobically oxidized to CO2, escapes into the atmosphere as greenhouse gas, or accumulates as methane hydrate in the deep sea.French press.MtrH was removed from the MtrABCDEFG(H) complex by chemical modification using dimethyl maleic anhydride (DMMA) and solubilized with 2.5% n-dodecyl-β-D-maltoside (DDM) as reported previously (11).The MtrABCDEFG complex was purified by DEAE-and Q-sepharose ion exchange chromatography and Superose-6 size exclusion chromatography.For grid preparation only protein of the peak fraction was applied.Detergent exchange was performed during the Qsepharose chromatography step by eluting with 50 mM MOPS/NaOH pH 7, 10 mM MgCl2, 2 mM DTT, 100-1000 mM NaCl and 3 CMC GDN (glyco-diosgenin).(B) Cryo-EM map (gray surface) and superimposed model at 2.37 Å resolution.The structures based on preparations with and without MtrH are virtually identical.The rms deviations are 0.3 Å.Despite differences in purification and detergent solubilization the found tetraether lipids and their positions remain nearly unchanged thereby documenting the high protein-lipid affinity.This also holds true for the different occupation of the two putative Na + .(C) The cryo-EM map after 3D classification.Using Relion (12) a map was calculated at 6.9 Å resolution based on 103947 particles without applying the threefold symmetry.
Three density pieces (one is behind the stalk) are visible above the noise level and might image the three MtrAs domains.(13,14), the most abundant tetraether glycolipid in M. marburgensis according to mass-spectrometric data (15).Additional methylations of the saturated isoprene chain and modifications of the polar heads (16,17) were not obvious in the density and thus not taken into accounts.Some of the lipids are well ordered (Fig. 1D), others adopt multiple conformations and their densities overlap with that of partner lipids.The terminal glycoside at the gentiobiosyl head is disordered in all lipids and was therefore not modelled.(B) Lipid binding sites in the Mtr(ABCDEFG)3 complex.Four of the 12 tetraether glycolipids (carbon in blue) are neighbored to each other thereby filling out the space between two MtrCDE globes (brown, yellow, red) and between the MtrBF and MtrAG helices of the stalk (green).The most interior tetraether glycolipid (see back arrow) is the one best defined (Fig. 1D).Tetraether lipids crossing the membrane in a rather natural manner are, to our knowledge, not characterized in the structures of membrane protein complexes but in hydrophobic clefts between tail and capsid of a spindle-shaped archaeal virus (18) and as substrate in tetraether lipid synthase (19).(C) Binding of a phosphoinositol head.The cytoplasmic surface of the membrane is characterized by positively charged residues (20) that form hydrogen bonds with the phosphate group of the lipids.The interacting ArgG50 is strictly conserved, ArgF41 is variable.The structural data indicate that lipids play a crucial role to connect different subunits and thus to increase the stability of the multi-modular complex.

Fig. S3 .
Fig. S3.MtrABCDEFGH of M. wolfeii.(A) The enzyme complex after sucrose gradient centrifugation.The pink color of the protein indicates the presence of MtrAs.(B) SDS-PAGE analysis after the final gel filtration step using a Superose TM6 Increase 10/300 GL column.(C) Liquid chromatography mass-spectrometric (LC-MS) analysis.For preparation, the gel bands were cut out, buffered and trypsin (SERVA) digested overnight.After acidification, the obtained peptides were applied to a C18 solid phase extraction column, eluted with 50% acetonitril/0.1% trifluoroacetic acid, dried and reconstituted in 0.1% trifluoroacetic acid.PSMs describes the total number of the identified peptide spectra that match the MtrABCDEFGH sequence.LC-MS data confirmed the presence of all eight subunits.The most frequently found subunits are MtrH and MtrA indicating that vitrification was started from a sample containing populations of the intact MtrABCDEFGH complex.(D) Cryo-EM map at 3.3 Å resolution.The cryo-EM density map (green surface) excellently fit with the superimposed model of the MtrAcBCDEFG complex.The soluble MtrAs domain and MtrH were not visible.The structures of MtrAcBCDEFG complex of M. marburgensis and M. wolfeii were virtually identical; the rms deviation is 0.43 Å by a sequence identity of 95%.

Fig. S4 .
Fig. S4.Biochemical analysis of the MtrABCDEFGH complex of M. marburgensis.(A) Elution profile of the final size exclusion chromatography.The protein complex was eluted after 13.5 ml retention volume from the Superose TM6 Increase 10/300 GL column.Only protein of the peak fraction was applied for grid preparation.The experiment was done 5 times with a yield of ca. 1 mg MtrABCDDEFGH from 30 g cells.Due to the limited amount of sample the B12 content and the Co oxidation state were not explicitly measured.We estimated the B12 content as rather high due to SDS-PAGE and mass-spectrometric data and the cob(III)amide portion as, unexpectedly, high due to the pink color and the high peak height at 365 nm relative to that at 280 nm.(B) SDS-PAGE after the final Superose TM6 gel filtration.Bands are highly similar in the M. marburgensis and M. wolfeii enzymes.

Fig. S5 .
Fig. S5.Cryo-EM Mtr(ABCDEFG)3 structure determination using Relion.(A) Workflow of data processing including a micrograph image, as well as 2D/3D classification and 3D refinement maps.Data collection was done on a Titan Krios EM at 300 kV equipped with a Gatan K3 detector using a pixel size of 0.837 Å at 105000x magnification.(B) Gold-standard FSC plot (green curve: unmasked; blue: masked; red, phase randomized masked; black, FSC corrected for overfitting).Resolution estimated at FSC=0.143.(C) Map of the protein complex viewed parallel to the membrane plane colored by local resolution.

Fig. S7 .
Fig. S7.Sequence alignment.Abbreviations used: M.marb, Methanothermobacter marburgensis; M.wolf, Methanothermobacter wolfeii, M.jann, Methanocaldococcus jannaschii; M.mari, Methanococcus maripaludis; M.bark, Methanosarcina barkeri; M.maze, Methanosarcina mazei; M.palu, Methanosphaerula palustris; M.hung, Methanospirillum hungatei; M.kand, Methanopyrus kandleri; M.okin, Methanothermococcus okinawensis; M.acet, Methanosarcina acetivorans; M.ther, M. thermolithotrophicus (A) MtrA.The significantly conserved linker between MtrAc and MtrAs is highlighted in green.Segments of MtrA close to the corrinoid ring and close to the B12 tail and those forming the interface to MtrH or MtrE (frequently overlapping) are shown in red, blue and orange, respectively.Residues forming the stalk interior are marked in magenta.(B) MtrB.Segment 7-15 (purple) forms an interface with MtrH according to Alphafold2.Invariant GlyB79 and GlyB87 (red) allow a close contact with MtrF.(C) MtrC, (D) MtrD, (E) MtrE.Regions contacting the MtrAs/corrinoid ring, CoM and Na + are marked in red, green and magenta, respectively.Helices II and V constituting the channel are drawn in brown.(F) MtrF.Invariant residues GlyF36 and GlyF44, involved in helix kinking, are marked in blue.No MtrF was found in M. maripaludis.(G) MtrG.The segment forming the interior of the stalk (magenta) is significantly conserved.M palustris and M. hungatei do not contain MtrG.(H) MtrH.Residues in contact and in neighborhood to H4MPT are shown in green.Residues close to MtrAs/corrinoid are marked in red; those contacting or close to MtrB are marked in violet and blue, respectively.

Fig. S9 .
Fig. S9.Contact area between the C-terminal region of MtrC and the residual protein complex.The C-terminal helix (226:252) of MtrC (orange) is strongly associated with helix IV (131:154) and the preceding loop of MtrE.The following cytoplasmic C-terminal arm (black) of MtrC interacts, in addition, with MtrB, MtrF and MtrG of the stalk.As described (Fig. S6C), the phosphoinositol heads of tetraether glycolipids (carbon in blue) also contribute to the interaction network between the subunits.

Fig. S10 .
Fig. S10.Cryo-EM Mtr(ABCDEFG)3-CoM structure determination using Relion.(A) Workflow of data processing including one image of a micrograph as well as 2D/3D classification and 3D refinement maps.(B) Gold-standard FSC plot (green curve: unmasked; blue: masked; red, phase randomized masked; black, FSC corrected for overfitting).Resolution estimated at FSC=0.143.(C) Map of the protein complex viewed parallel to the membrane plane colored by local resolution.

Fig. S11 .
Fig. S11.Density modified map at the hydrophilic Na + binding pocket.One metal binding site could be clearly detected according to the high quality of the density at 1.99 Å resolution and metal-ligand distances.The second potential metal binding site is only weakly occupied and cannot be definitively discriminated from a binding site of a firmly bound water molecule.A further attractive hydrophilic pocket lies adjacent to the conserved GluE28, GlnE34 and LysE56 but density for a metal was not visible.A further metal binding site was tentatively identified at the extracellular boundary between AlaB97-O, LeuB100-OXT and LeuA'235-O and three protein-linked solvent molecules involved in the fixation of different subunits.A Zn 2+ binding site present in other thiol-activating methyltransferases(22,23) or even nearby positioned potential ligands as cysteine, histidine and glutamate/aspartate were not found adjacent to CoM, although space is available in the oversized cavity.

Fig. S12 .
Fig. S12.Alphafold2 model of the MtrH dimer (dark-gray, light-gray) of M. marburgensis.The Alphafold2 structures of MtrH of M. marburgensis, M. wolfeii, M. kandleri, M. barkeri, M. jannaschii and M. maripaludis are virtually identical.The MtrH monomer reveals the well-known TIM barrel architecture that notably deviates from the classical fold by two antiparallel β-strands attached to the first TIM barrel strand covering the bottom of the TIM barrel and two helices downstream the prolonged helix H247:H262 involved in oligomerization.The MtrH dimer is superimposed with theMtgA dimer (tomato, orange) of D. hafniense determined in complex with methyl-H4F (carbon in turquoise)(24).MtrH and MtgA rms deviate 1.9 Å (301 from 304 residues; 35% sequence identity; TM: 0.91).The high conservation of their monomer-monomer interfaces strongly argues for a homodimeric MtrH.Experimental hints about a homodimeric state of MtrH were obtained, when purifying MtrH of M. marburgensis from the membrane protein fraction after separating MtrH from the MtrABCDEFGH complex with DMMA(11).MtgA is involved in the N-demethylation of the quaternary amine glycine betaine, which is used for energy production and carbon assimilation in acetogenic bacteria.It catalyzes the methyl transfer from methyl-cob(III)alamin to tetrahydrofolate.

Fig. S13 .
Fig. S13.Alphafold2 model of MtrA from M. marburgensis.MtrA (blue) adopts a Rossmann fold and is nearly identical with the MtrA homolog of M. fervidus (tan) (29) reflected in a rms deviation of 0.49 Å (160 of 163 residues; TM: 0.82).B12 (pink) was transferred into M. marburgensis MtrAs with minorside chain adjustments to avoid a collision.The exposed corrinoid interacts with loops after strands A29:A31, A47:A50 and A73:A77 as well as segment A111-A112.As described previously, the binding mode of B12 is rather different in MtrA compared to most other B12-containing proteins(29) despite the common Rossmann fold scaffold(30).HisA84 was identified as the Co ligand by sitedirected mutagenesis(31) and confirmed by structural data(29).The segment A80-A115 (dark-blue) including helix A85:A94 is perhaps strongly influenced by the position of HisA84, which in turn depends on whether Co is in the square-planar Co(I) and the octahedral CH3-Co(III)(His-on) states.HisA84-NE1 is hydrogen-bonded with GluA54-OE1; the latter interacts with GluA54N and AlaA111N.

Fig. S14 .
Fig. S14.Quality analysis of Alphafold2 calculations (32) of the MtrAs-MtrH and MtrA-MtrCDE models.(A) pLDDT (predicted local distance difference test) coloring (blue to yellow) representation of the MtrAs-MtrH subcomplex from M. marburgensis.The pLDDT values of the MtrH dimer andMtrAs are very high and the prediction reliable, but those of the contact region are relatively low (orange-green) and cannot be applied as single information source.However, a similar interface was obtained by independent calculations for the M. marburgensis, M. thermoautotrophicum, M. wolfeii, M. mazei, M. jannaschii and M. hungatei MtrAs-MtrH subcomplexes.Moreover, the evaluation of these results has to consider that the exposed corrinoid of MtrAs (but also methyl-H4MPT of MtrH) is not included in the Alphafold2 calculations.However, the corrinoid forms a significant fraction of the contact area and also influences the conformation of surrounding segments of MtrAs that also partly form the interface to MtrH (Fig.5A).(B) pLDDT coloring (blue to yellow) representation of the MtrA-MtrCDE subcomplex.The pLDDT values of residues of the interface region are rather poor.Again, B12 is a major component of the contact area (Fig.5B).Proteins from M. marburgensis, M. wolfeii, M. jannaschii, M. kandleri, M. barkeri and M. maripaludis revealed a similar interface.The conformation of the MtrA linker is highly undefined according to Alphafold2 calculations, which reflects its function.The position of the C-terminal MtrA helix MtrAc cannot be assessed, as the other stalk helices were not included in the calculation.

Fig. S15 .
Fig. S15.Cartoon representation of the Mtr(ABCDEFG)3H2 complex in the conformation of the two half-reactions.(A) The methyl-H4MPT demethylation state.The model is composed of the cryo-EM Mtr(AcBCDEFG)3 complex, the MtrBH2 module from the superimposed Alphafold2 model Mtr(AcBFG)3H2 (MtrAcBFG green, MtrH monomers dark-gray and bright-gray) and MtrAs (blue) of the superimposed MtrAsH2 model (Fig. 5A).The visible subunits are underlined.MtrH2 predominantly forms an interface with MtrB but MtrF and MtrG might also contribute.Alphafold2 models of the MtrB-MtrH2 subcomplex calculated from M. marburgensis, M. wolfeii, M. jannaschii, M. kandleri, M. barkeri and M. maripaludis sequences are virtually identical.As presented in the inset, the N-terminal 25 amino acids of MtrB, which are disordered in the cryo-EM map fold as a flat three-stranded antiparallel β-sheet (green) localized at the top of the stalk.The three β-strands are placed into the cleft formed between the two MtrH subunits such that the N-terminal residues H3-H5 of the latter add one strand each (magenta) to constitute a five-stranded mixed β-sheet.Moreover, an extended hydrophobic patch is formed between ValB7, IleB9, LeuB17, IleB24, PheH4, PheH213, PheH2, PheH'2 (the prime describes the second monomer of MtrH2), PheH4' and PheH'213.LysB8 and AspH50 are linked by a salt bridge.In the presented Mtr(ABCDEFG)3H2 model the MtrA linker is rather strained but it is conceivable that MtrB-MtrH2 might be more inclined towards the membrane thereby shortening the distance to the stalk and relaxing the MtrA linker.Residues AlaB27-GlyB29 belong neither to the threestranded N-terminal β-sheet nor to the MtrB part of the stalk visible in the cryo-EM map and may act as a flexible linker.If this holds true, the three-stranded N-terminal β-sheet of MtrB (B1-B26) and the associated MtrH2 are mobile.Thus, MtrH might be part of the vitrified protein complex used for cryo-